Method for in situ inhibition of regulatory t cells

ABSTRACT

The present invention pertains to engineered T-cells, method for their preparation and their use as medicament, particularly for immunotherapy. The engineered T-cells of the invention are designed to express both a Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell, and a secreted inhibitor of regulatory T-cells (Treg). Preferably, such secreted inhibitor is a peptide inhibitor of forkhead/winged helix transcription factor 3 (FoxP3), a specific factor involved into the differentiation of T-cells into regulatory T-cells. The engineered T-cells of the invention direct their immune activity towards specific malignant or infected cells, while at the same time will prevent neighbouring regulatory T-cells from modulating the immune response. The invention opens the way to standard and affordable adoptive immunotherapy strategies, especially for treating or preventing cancer, and bacterial or viral infections.

FIELD OF THE INVENTION

The present invention pertains to engineered T-cells, method for theirpreparation and their use as medicament, particularly for immunotherapy.The engineered T-cells of the invention are designed to express locallya secreted inhibitor of regulatory T-cells (Treg). Preferably, suchsecreted inhibitor is a peptide inhibitor of forkhead/winged helixtranscription factor 3 (FoxP3), a specific factor involved into thedifferentiation of T-cells into regulatory T-cells. Said T-cells arepreferably endowed with Chimeric Antigen Receptors (CAR) directedagainst at least one antigen expressed at the surface of a malignant orinfected cell. The engineered T-cells of the invention thereby directtheir immune activity towards specific malignant or infected cells,while at the same time prevent neighbouring regulatory T-cells frommodulating the immune response. The invention opens the way to standardand affordable adoptive immunotherapy strategies, especially fortreating or preventing cancer, and bacterial or viral infections.

BACKGROUND OF THE INVENTION

Cellular adaptive immunity is mediated by T-lymphocytes, also known asT-cells, which upon recognition of a non-self or tumoral antigen caneither destroy the target cell or orchestrate an immune response withother cells of the immune system.

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T-cells generated ex vivo, is a promising strategy totreat viral infections and cancer. The T-cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specificT-cells or redirection of T-cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T-cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T-cells has beenshown to be successful in treating melanoma.

Novel specificities in T-cells have been successfully generated throughthe genetic transfer of transgenic T-cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T-cells.CARs have successfully allowed T-cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

While cytotoxic T-lymphocyte (CTL; also known as cytotoxic T-cells) andT helper cells play a central role in the cellular immune response,regulatory T-cells (Tregs), formerly known as suppressor T-cells,modulate or suppress immune responses, particularly to preventautoimmunity and maintain tolerance to self-antigen. Because of theirimmune regulatory function, the presence of regulatory T-cells at acancer or infection site may hinder the induction of an immune responseagainst cancer or infectious pathogens (Aandahl E. M. et al. (2004);Cabrera R. et al. (2004); Viguier M. et al. (2004); Woo E. Y. et al(2001)). Therefore, in certain pathogenic situations such as chronicinfectious diseases or cancer it may be desirable to suppress theactivity of regulatory T-cells to allow a more potent immune response tooccur. On another hand, vaccine strategies were developed based on thefinding that vaccine efficacy could be improved by reducing the activityof regulatory T-cells, for instance by controlling the activity of theforkhead/winged helix transcription factor 3 (FoxP3). In particular, apeptide inhibitor of FOXP3, called P60, was found to improve vaccineefficacy in mice (Casares et al., 2010).

Dysfunction of FOXP3 is however associated with serious autoimmunedisorders such as systemic lupus erythematosus or X-lined IPEX syndrome,such that systemic administration of inhibitors directed against, e.g,FoxP3, is currently not deemed a suitable option in immune therapy. Therelease of such inhibitors in the blood stream or even locally may leadto toxic effects by unleashing autoimmune reactions in organs notaffected by the primary disease.

Accordingly, new therapeutic strategies are needed to facilitate aneffective immune response while reducing possible toxic side effects innon-affected areas of the body. This need is addressed by the presentinvention by providing specific in-situ inhibition of regulatory T-cellsas part of a CAR immunotherapy.

SUMMARY OF THE INVENTION

The present invention concerns methods for preparing engineered T-cellsable to neutralize the activity of regulatory T-cells in the closeenvironment of pathological cells by heterologous expression of a factorinhibiting the activity of said regulatory T-cells.

According to one aspect of the invention, the method can be moreparticularly applied to tumor-infiltrating lymphocytes (TIL), which areT-cells expressing endogenous antigen receptor specific for tumor cells(i.e. upon natural clonal selection within the patient). According tothis aspect of the invention, the cells are relieved from theirinhibition by regulatory T-cells to help their spread and action againstpatient's tumor cells. Particularly, TIL are extracted from a patient'stumor, amplified, genetically modified to express an inhibitor ofregulatory T-cells, and then re-introduced into the patient as atherapeutic product. Such TIL engineered ex-vivo can be used to treatthe original patient (autologous strategy) or another patient who bearsthe same type of tumor (allogeneic approach). Such method moreparticularly comprises one or several of the steps of:

-   -   a) extracting a tumor-infiltrating lymphocyte from a patient's        tumor;    -   b) expanding said tumor-infiltrating lymphocyte; and    -   c) introducing into said tumor-infiltrating lymphocyte an        exogenous nucleic acid molecule comprising a nucleotide sequence        coding for an inhibitor of regulatory T-cell activity.

According to another aspect of the invention, the method can be moreparticularly applied to T-cells interacting with the pathological cellsthrough a chimeric antigen receptor. Such method more particularlycomprises one or several of the steps of:

a) providing a T-cell;

b) introducing into said T-cell an exogenous nucleic acid moleculecomprising a nucleotide sequence coding for a first Chimeric AntigenReceptor (CAR) directed against at least one antigen expressed at thesurface of a malignant or infected cell; and

c) introducing into said T-cell an exogenous nucleic acid moleculecomprising a nucleotide sequence coding for an inhibitor of regulatoryT-cells activity.

By “inhibitor of regulatory T-cells” is meant a molecule or precursor ofsaid molecule secreted by the T-cells and which allow T-cells to escapethe down regulation activity exercised by the regulatory T-cellsthereon. In general, such inhibitor of regulatory T-cell activity hasthe effect of reducing FoxP3 transcriptional activity in said cells.

According to preferred embodiments, said inhibitor of regulatory T-cellactivity is an inhibitor of FoxP3, and more preferably is acell-penetrating peptide inhibitor of FoxP3, such as that referred asP60 (Casares et al., 2010).

According to preferred embodiments, the engineered T-cell concomitantlyexpresses a CAR on its surface that binds a surface antigen marker of apathological cell. This binding has the effect of triggering an immuneresponse by the T-cell directed against the pathological cell, whichresult into degranulation of various cytokine and degradation enzymes inthe interspace between the cells.

According to certain embodiments, the method comprises a further step ofintroducing into said T-cell an exogenous nucleic acid moleculecomprising a nucleotide sequence coding for a second Chimeric AntigenReceptor directed against at least one antigen expressed at the surfaceof a regulatory T-cell (Treg). After having introduced said nucleicacid, said second Chimeric Antigen Receptor may then be expressed bysaid T-cell.

Said second CAR is directed against the regulatory T-cells in order toprimarily physically maintain said regulatory T-cells in the closeenvironment of the T-cells (and also of the pathological cells) forobtaining in-situ inhibition of the regulatory T-cells. The second CARcan also contribute to activating the T-cells immune response.

According to optional embodiments, the method further comprises the stepof making the T-cells non-alloreactive by inactivating at least one genecoding for one component of the T-Cell receptor (TCR). This can beachieved by introducing into the cells a specific rare-cuttingendonuclease targeting this gene, such as a TAL-nuclease, a CAS9 RNAguided endonuclease, a Zinc Finger nuclease or a meganuclease.

The present invention thus preferably provides engineered T-cells, inparticular. genetically engineered isolated T-cells comprising:

a) an exogenous nucleic acid molecule comprising a nucleotide sequencecoding for a first Chimeric Antigen Receptor (CAR) directed against atleast one antigen expressed at the surface of a malignant or infectedcell; and

b) an exogenous nucleic acid molecule comprising a nucleotide sequencecoding for an inhibitor of regulatory T-cell activity, preferably acell-penetrating peptide inhibitor of FoxP3, such as that referred asP60 (Casares et al., 2010).

According to preferred embodiments, said first Chimeric Antigen Receptorand said inhibitor of regulatory T-cell activity are expressed by saidT-cell.

According to other embodiments, the engineered T-cell further comprisesc) an exogenous nucleic acid molecule comprising a nucleotide sequencecoding for a second Chimeric Antigen Receptor directed against at leastone antigen expressed at the surface of a regulatory T-cell (Treg).According to particular embodiments, said second Chimeric AntigenReceptor is expressed by said T-cell.

According to further embodiments, the engineered T-cell furthercomprises d) an exogenous nucleic acid molecule comprising a nucleotidesequence coding for a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage at least one gene coding for one component ofthe T-Cell receptor (TCR). According to particular embodiments, saidrare-cutting endonuclease able to selectively inactivate by DNA cleavageat least one gene coding for one component of the T-Cell receptor (TCR)is expressed by said T-cell. The disruption of TCR providesnon-alloreactive T-cells that can be used in allogeneic treatmentstrategies.

The present invention further provides isolated nucleic acid moleculescomprising a nucleotide sequence coding for a Chimeric Antigen Receptor(CAR) directed against at least one antigen expressed at the surface ofa malignant or infected cell; and a nucleotide sequence coding for aninhibitor of regulatory T-cell activity, preferably a cell-penetratingpeptide inhibitor of FoxP3, such as that referred as P60 (Casares etal., 2010). According to certain embodiments, said nucleic acid moleculeis a vector, such as a viral vector or plasmid. More particularly, saidnucleic acid molecule is a vector, such as a viral vector or plasmid,and said nucleotide sequences being operatively linked to one or morepromoters suitable for expression in a T-cell.

The present invention further provides compositions comprising one ormore nucleic acid molecules comprising a nucleotide sequence coding fora first Chimeric Antigen Receptor (CAR) directed against at least oneantigen expressed at the surface of a malignant or infected cell; and anucleotide sequence coding for an inhibitor of regulatory T-cellactivity, preferably a cell-penetrating peptide inhibitor of FoxP3, suchas that referred as P60 (Casares et al., 2010). According to certainembodiments, the composition comprises a nucleic acid moleculecomprising a nucleotide sequence coding for said first Chimeric AntigenReceptor (CAR); and a nucleotide sequence coding for said an inhibitorof regulatory T-cell activity. According to certain other embodiments,the composition comprises a first nucleic acid molecule comprising anucleotide sequence coding for said first Chimeric Antigen Receptor(CAR) directed against at least one antigen expressed at the surface ofa malignant or infected cell; and a second nucleic acid moleculecomprising a nucleotide sequence coding for an inhibitor of regulatoryT-cell activity, preferably a cell-penetrating peptide inhibitor ofFoxP3, such as that referred as P60 (Casares et al., 2010). According tocertain embodiments, the composition comprises a further nucleic acidmolecule comprising a nucleotide sequence coding for a second ChimericAntigen Receptor directed against at least one antigen expressed at thesurface of a regulatory T-cell (Treg). According to certain embodiments,said nucleic acid molecules are vectors, such as viral vectors orplasmids. More particularly, said nucleic acid molecules are vectors,such as viral vectors or plasmids, and said nucleotide sequences beingoperatively linked to one or more promoters suitable for expression in aT-cell.

The present invention further provides kits comprising one or moreisolated nucleic acid according to the present invention or one or morecompositions according to the present invention.

As a result of the present invention, engineered T-cells can be used astherapeutic products, ideally as an “off the shelf” product, for use inthe treatment or prevention cancer, bacterial or viral infections, orauto-immune diseases. Thus, the present invention further provides anengineered T-cell or a composition, such as a pharmaceuticalcomposition, comprising same for use as a medicament. According tocertain embodiments, the engineered T-cell or composition is for use inthe treatment of a cancer, and more particularly for use in thetreatment of lymphoma. According to certain other embodiments, theengineered T-cell or composition is for use in the treatment of viralinfection. According to certain other embodiments, the engineered T-cellor composition is for use in the treatment of bacterial infection.

It is understood that the details given herein with respect to oneaspect of the invention also apply to any of the other aspects of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the engineered T-cell according theinvention expressing both a Chimeric Antigen Receptor (CAR) directedagainst at least one antigen expressed at the surface of a malignant orinfected cell, and a cell-penetrating peptide inhibitor of FoxP3. Thepeptide inhibitor of FoxP3 expressed and secreted by the engineeredT-cell will enter neighbouring regulatory T-cells and prevent them frommodulating the anti-tumor or anti-infection response.

FIG. 2: Schematic representation of an engineered T-cell according theinvention expressing a Chimeric Antigen Receptor (CAR) directed againstat least one antigen expressed at the surface of a malignant or infectedcell, and a cell-penetrating peptide inhibitor of FoxP3 as well as arare-cutting endonuclease able to selectively inactivate by DNA cleavageat least one gene coding for one component of the T-Cell receptor (TCR).The inactivation of at least one gene coding for a TCR component rendersthe genetically engineered T-cell non-alloreactive.

FIG. 3: Schematic representation of an engineered T-cell according theinvention expressing a first Chimeric Antigen Receptor directed againstat least one antigen expressed at the surface of a malignant or infectedcell, a cell-penetrating peptide inhibitor of FoxP3 as well as a secondChimeric Antigen Receptor directed against at least one antigenexpressed at the surface of a regulatory T-cell (Treg) which allows thebinding of a regulatory T-cell by the engineered T-cell of the inventionand facilitates the entry of the peptide inhibitor of FoxP3 into theregulatory T-cell.

FIG. 4: Schematic representation of an engineered T-cell according theinvention expressing a first Chimeric Antigen a first Chimeric AntigenReceptor directed against at least one antigen expressed at the surfaceof a malignant or infected cell, a cell-penetrating peptide inhibitor ofFoxP3 as well as a second Chimeric Antigen Receptor directed against atleast one antigen expressed at the surface of a regulatory T-cell(Treg). In this configuration, the engineered T-cell does notnecessarily express a transgene that codes for an inhibitor of Treg, butcan neutralize Treg by merely specifically binding to them (e.g. CD25 isa specific Treg surface protein).

FIG. 5: Schematic representation of an engineered T-cell expressing afirst Chimeric Antigen a first Chimeric Antigen Receptor, whichcytotoxic activity is inhibited by a Treg.

FIG. 6: Schematic representation of an engineered T-cell according tothe invention expressing a first Chimeric Antigen a first ChimericAntigen Receptor and overexpressing p60 as a cell-penetrating peptideinhibitor of FoxP3 with the effect that the inhibition by the Treg islifted and the T cells recovers cytolytic activity against the tumorcells.

DETAILED DESCRIPTION OF THE INVENTION

Unless specifically defined herein, all technical and scientific termsused have the same meaning as commonly understood by a skilled artisanin the fields of gene therapy, biochemistry, genetics, and molecularbiology.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,with suitable methods and materials being described herein. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willprevail. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, CurrentProtocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley andson Inc, Library of Congress, USA); Molecular Cloning: A LaboratoryManual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J.Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Harries & S. J. Higgins eds. 1984); TranscriptionAnd Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture OfAnimal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); ImmobilizedCells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide ToMolecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelsonand M. Simon, eds.-in-chief, Academic Press, Inc., New York),specifically, Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “GeneExpression Technology” (D. Goeddel, ed.); Gene Transfer Vectors ForMammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold SpringHarbor Laboratory); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

Methods for Preparing Engineered T-Cells

In a general aspect, the present invention pertains to methods forpreparing engineered T-cells that have the ability to lift theirinhibition by Treg, preferably by heterologous expression of aninhibitor of Treg, such as a penetrating peptide inhibitor of FoxP3.

Because regulatory T-cells, also known as suppressor T-cells, play arole in dampening immune responses, in particular to preventautoimmunity and maintaining tolerance of self-antigens, it is desirableto suppress the activity of this cell type in certain pathogenicsituations, such as cancer or chronic infectious diseases, to allow amore potent immune response to occur. In order to allow a localsuppression of regulatory T-cells, an inhibitor is secreted by theengineered T-cell, preferably a peptide inhibitor of FoxP3 into theenvironment of the engineered T-cell(s). This later peptide inhibitorwill enter neighbouring regulatory T-cells and prevent them frommodulating the immune response by inhibition of FoxP3. The localizeddelivery of the peptide inhibitor of FoxP3 by the engineered T-cell(s)of the present invention has the great advantage of reducing thepossibility of toxic effects such inhibitor would unfold elsewhere inthe body.

According to a first aspect, the invention is applied to a subset ofT-cells called tumor-infiltrating lymphocytes (TIL), which are found intumors with the particularity of having developed some affinity to atleast a population of tumor cells found in said tumors. Generally, theseTILs remain active against the tumor cells and thus are valuable inimmunotherapy for treating said tumors (Rosenberg, S A. et al. (1986) Anew approach to the adoptive immunotherapy of cancer withtumor-infiltrating lymphocytes. Science. 233(4770):1318-1321). However,these cells are generally in limited number and are confined to suchtumors. The invention allows enhancing their activities and helpingtheir proliferation by proceedings by one or several of the followingsteps:

-   -   Extracting TILs from one or several tumors from one or several        patients;    -   Expanding the TILs to obtain a significant number useful for        immunotherapy, preferably up to at least 105 cells, more        preferably up to at least 106 cells;    -   Introducing into the cells a nucleic acid comprising a        nucleotide sequence coding for an inhibitor of regulatory T-cell        activity;    -   Further activating and expanding the engineered TILs; and    -   Infusing the engineered TILs back into the patient or in other        patients.

According to another aspect, the T-cells are engineered to express botha Chimeric Antigen Receptor (CAR) directed against at least one antigenexpressed at the surface of a malignant or infected cell, herein denotedfirst CAR, and an inhibitor of regulatory T-cell activity, inparticular, a cell-penetrating peptide inhibitor of FoxP3. The CAR willdirect the engineered T-cells to the tumor site or site of infection andallows the T-cell(s) to kill the tumor or infected cells.

Accordingly, the present invention provides a method for preparing anengineered T-cell comprising the steps of:

a) providing a T-cell;

b) introducing into said T-cell an exogenous nucleic acid moleculecomprising a nucleotide sequence coding for a first Chimeric AntigenReceptor (CAR) directed against at least one antigen expressed at thesurface of a malignant or infected cell; and

c) introducing into said T-cell an exogenous nucleic acid moleculecomprising a nucleotide sequence coding for an inhibitor of regulatoryT-cell activity, such as a cell-penetrating peptide inhibitor of FoxP3.

As a result, an engineered T-cell is obtained which expresses a firstChimeric Antigen Receptor (CAR) directed against at least one antigenexpressed at the surface of a malignant or infected cell, and aninhibitor of regulatory T-cell activity, such as a cell-penetratingpeptide inhibitor of FoxP3.

In addition to the Chimeric Antigen Receptor (CAR) directed against atleast one antigen expressed at the surface of a malignant or infectedcell, it may be preferable to have a further CAR expressed by theengineered T-cell which is directed against at least one antigenexpressed at the surface of a regulatory T-cell (Treg), such as thesurface antigen CD25. This will allow the binding of a regulatory T-celland facilitates the entry of the inhibitor of regulatory T-cellactivity. Accordingly, the method may further comprise introducing intosaid T-cell an exogenous nucleic acid molecule comprising a nucleotidesequence coding for a second Chimeric Antigen Receptor directed againstat least one antigen expressed at the surface of a regulatory T-cell(Treg). After having introduced said nucleic acid, said second ChimericAntigen Receptor may then be expressed by said T-cell.

As a result, an engineered T-cell is obtained which further expresses asecond Chimeric Antigen Receptor directed against at least one antigenexpressed at the surface of a regulatory T-cell (Treg).

It is also contemplated by the present invention that the engineeredT-cell of the present invention further expresses a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage at least onegene coding for one component of the T-Cell receptor (TCR). T-cellreceptors are cell surface receptors that participate in the activationof T cells in response to the presentation of antigen. The TCR isgenerally made from two chains, alpha and beta, which assemble to form aheterodimer and associates with the CD3-transducing subunits to form theT-cell receptor complex present on the cell surface. Each alpha and betachain of the TCR consists of an immunoglobulin-like N-terminal variable(V) and constant (C) region, a hydrophobic transmembrane domain, and ashort cytoplasmic region. As for immunoglobulin molecules, the variableregion of the alpha and beta chains are generated by V(D)Jrecombination, creating a large diversity of antigen specificitieswithin the population of T cells. However, in contrast toimmunoglobulins that recognize intact antigen, T cells are activated byprocessed peptide fragments in association with an MHC molecule,introducing an extra dimension to antigen recognition by T cells, knownas MHC restriction. Recognition of MHC disparities between the donor andrecipient through the T cell receptor leads to T cell proliferation andthe potential development of graft versus host disease (GVHD). It hasbeen shown that normal surface expression of the TCR depends on thecoordinated synthesis and assembly of all seven components of thecomplex (Ashwell and Klusner 1990). The inactivation of TCRalpha orTCRbeta can result in the elimination of the TCR from the surface of Tcells preventing recognition of alloantigen and thus GVHD. Theinactivation of at least one gene coding for a TCR component thusrenders the genetically engineered T-cell non-alloreactive. By“inactivating” or “inactivation of” a gene it is meant that the gene ofinterest is not expressed in a functional protein form.

Accordingly, the method of the present invention may further compriseintroducing into said T-cell an exogenous nucleic acid moleculecomprising a nucleotide sequence coding for a rare-cutting endonucleaseable to selectively inactivate by DNA cleavage, preferably double-strandbreak, at least one gene coding for one component of the T-Cell receptor(TCR). In particular embodiments, the rare-cutting endonuclease is ableto selectively inactivate by DNA cleavage the gene coding for TCR alphaor TCR beta.

The term “rare-cutting endonuclease” refers to a wild type or variantenzyme capable of catalyzing the hydrolysis (cleavage) of bonds betweennucleic acids within a DNA or RNA molecule, preferably a DNA molecule.Particularly, said nuclease can be an endonuclease, more preferably arare-cutting endonuclease which is highly specific, recognizing nucleicacid target sites ranging from 10 to 45 base pairs (bp) in length,usually ranging from 10 to 35 base pairs in length, more usually from 12to 20 base pairs. The endonuclease according to the present inventionrecognizes at specific polynucleotide sequences, further referred to as“target sequence” and cleaves nucleic acid inside these target sequencesor into sequences adjacent thereto, depending on the molecular structureof said endonuclease. The rare-cutting endonuclease can recognize andgenerate a single- or double-strand break at specific polynucleotidessequences.

In a particular embodiment, said rare-cutting endonuclease according tothe present invention is a RNA-guided endonuclease such as theCas9/CRISPR complex. RNA guided endonucleases constitute a newgeneration of genome engineering tool where an endonuclease associateswith a RNA molecule. In this system, the RNA molecule nucleotidesequence determines the target specificity and activates theendonuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al.2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013). Cas9, also namedCsn1 is a large protein that participates in both crRNA biogenesis andin the destruction of invading DNA. Cas9 has been described in differentbacterial species such as S. thermophiles, Listeria innocua (Gasiunas,Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and S. pyogenes(Deltcheva, Chylinski et al. 2011). The large Cas9 protein (>1200 aminoacids) contains two predicted nuclease domains, namely HNH (McrA-like)nuclease domain that is located in the middle of the protein and asplitted RuvC-like nuclease domain (RNase H fold). Cas9 variant can be aCas9 endonuclease that does not naturally exist in nature and that isobtained by protein engineering or by random mutagenesis. Cas9 variantsaccording to the invention can for example be obtained by mutations i.e.deletions from, or insertions or substitutions of at least one residuein the amino acid sequence of a S. pyogenes Cas9 endonuclease (COG3513).

In a particular embodiment, said rare-cutting endonuclease can also be ahoming endonuclease, also known under the name of meganuclease. Suchhoming endonucleases are well-known to the art (Stoddard 2005). Homingendonucleases are highly specific, recognizing DNA target sites rangingfrom 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40bp in length. The homing endonuclease according to the invention may forexample correspond to a LAGLIDADG endonuclease, to a HNH endonuclease,or to a GIY-YIG endonuclease. Preferred homing endonuclease according tothe present invention can be an I-CreI variant. A “variant”endonuclease, i.e. an endonuclease that does not naturally exist innature and that is obtained by genetic engineering or by randommutagenesis can bind DNA sequences different from that recognized bywild-type endonucleases (see international application WO2006/097854).

In a particular embodiment, said rare-cutting endonuclease can be a“Zinc Finger Nucleases” (ZFNs), which are generally a fusion between thecleavage domain of the type IIS restriction enzyme, FokI, and a DNArecognition domain containing 3 or more C2H2 zinc finger motifs. Theheterodimerization at a particular position in the DNA of two individualZFNs in precise orientation and spacing leads to a double-strand break(DSB) in the DNA. The use of such chimeric endonucleases have beenextensively reported in the art as reviewed by Urnov et al. (Genomeediting with engineered zinc finger nucleases (2010) Nature reviewsGenetics 11:636-646). Standard ZFNs fuse the cleavage domain to theC-terminus of each zinc finger domain. In order to allow the twocleavage domains to dimerize and cleave DNA, the two individual ZFNsbind opposite strands of DNA with their C-termini a certain distanceapart. The most commonly used linker sequences between the zinc fingerdomain and the cleavage domain requires the 5′ edge of each binding siteto be separated by 5 to 7 bp. The most straightforward method togenerate new zinc-finger arrays is to combine smaller zinc-finger“modules” of known specificity. The most common modular assembly processinvolves combining three separate zinc fingers that can each recognize a3 base pair DNA sequence to generate a 3-finger array that can recognizea 9 base pair target site. Numerous selection methods have been used togenerate zinc-finger arrays capable of targeting desired sequences.Initial selection efforts utilized phage display to select proteins thatbound a given DNA target from a large pool of partially randomizedzinc-finger arrays. More recent efforts have utilized yeast one-hybridsystems, bacterial one-hybrid and two-hybrid systems, and mammaliancells.

In a particular embodiment, said rare-cutting endonuclease is a“TALE-nuclease” or a “MBBBD-nuclease” resulting from the fusion of a DNAbinding domain typically derived from Transcription Activator LikeEffector proteins (TALE) or from a Modular Base-per-Base Binding domain(MBBBD), with a catalytic domain having endonuclease activity. Suchcatalytic domain usually comes from enzymes, such as for instanceI-TevI, ColE7, NucA and Fok-I. TALE-nuclease can be formed undermonomeric or dimeric forms depending of the selected catalytic domain(WO2012138927). Such engineered TALE-nucleases are commerciallyavailable under the trade name TALEN™ (Cellectis, 8 rue de la CroixJarry, 75013 Paris, France). In general, the DNA binding domain isderived from a Transcription Activator like Effector (TALE), whereinsequence specificity is driven by a series of 33-35 amino acids repeatsoriginating from Xanthomonas or Ralstonia bacterial proteins AvrBs3,PthXo1, AvrHah1, PthA, Tal1c as non-limiting examples. These repeatsdiffer essentially by two amino acids positions that specify aninteraction with a base pair (Boch, Scholze et al. 2009; Moscou andBogdanove 2009). Each base pair in the DNA target is contacted by asingle repeat, with the specificity resulting from the two variant aminoacids of the repeat (the so-called repeat variable dipeptide, RVD). TALEbinding domains may further comprise an N-terminal translocation domainresponsible for the requirement of a first thymine base (T0) of thetargeted sequence and a C-terminal domain that containing a nuclearlocalization signals (NLS). A TALE nucleic acid binding domain generallycorresponds to an engineered core TALE scaffold comprising a pluralityof TALE repeat sequences, each repeat comprising a RVD specific to eachnucleotides base of a TALE recognition site. In the present invention,each TALE repeat sequence of said core scaffold is made of 30 to 42amino acids, more preferably 33 or 34 wherein two critical amino acids(the so-called repeat variable dipeptide, RVD) located at positions 12and 13 mediates the recognition of one nucleotide of said TALE bindingsite sequence; equivalent two critical amino acids can be located atpositions other than 12 and 13 specially in TALE repeat sequence tallerthan 33 or 34 amino acids long. Preferably, RVDs associated withrecognition of the different nucleotides are HD for recognizing C, NGfor recognizing T, NI for recognizing A, NN for recognizing G or A. Inanother embodiment, critical amino acids 12 and 13 can be mutatedtowards other amino acid residues in order to modulate their specificitytowards nucleotides A, T, C and G and in particular to enhance thisspecificity. A TALE nucleic acid binding domain usually comprisesbetween 8 and 30 TALE repeat sequences. More preferably, said corescaffold of the present invention comprises between 8 and 20 TALE repeatsequences; again more preferably 15 TALE repeat sequences. It can alsocomprise an additional single truncated TALE repeat sequence made of 20amino acids located at the C-terminus of said set of TALE repeatsequences, i.e. an additional C-terminal half-TALE repeat sequence.Other modular base-per-base specific nucleic acid binding domains(MBBBD) are described in WO 2014/018601. Said MBBBD can be engineered,for instance, from newly identified proteins, namely EAV36_BURRH,E5AW43_BURRH, E5AW45_BURRH and E5AW46_BURRH proteins from the recentlysequenced genome of the endosymbiont fungi Burkholderia Rhizoxinica.These nucleic acid binding polypeptides comprise modules of about 31 to33 amino acids that are base specific. These modules display less than40 sequence identity with Xanthomonas TALE common repeats and presentmore polypeptides sequence variability. The different domains from theabove proteins (modules, N and C-terminals) from Burkholderia andXanthomonas are useful to engineer new proteins or scaffolds havingbinding properties to specific nucleic acid sequences and may becombined to form chimeric TALE-MBBBD proteins. As a result, anengineered T-cell is obtained which further expresses a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage at least onegene coding for one component of the T-Cell receptor (TCR).

It is also contemplated by the present invention that the engineeredT-cell of the present invention further expresses a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage, preferablydouble-strand break, the gene coding for the surface antigen CD25. Byinactivating the gene coding for CD25 the engineered T-cells areunlikely to self-associate or to self-interact and prevented fromtargeting T-cells other than Treg.

The T-cell to be modified according to the present invention may be anysuitable T-cell. For example, the T-cell can be an inflammatoryT-lymphocyte, cytotoxic T-lymphocyte, or helper T-lymphocyte.Particularly, the T-cell is a cytotoxic T-lymphocyte. In certainembodiments, said T-cell is selected from CD4+ T-lymphocytes and CD8+T-lymphocytes. In particular embodiments, the T-cell to be modifiedaccording to the present invention is a human T-cell. Prior to expansionand genetic modification of the cells of the invention, a source ofcells can be obtained from a subject, such as a patient, through avariety of non-limiting methods. T-cell can be obtained from a number ofnon-limiting sources, including peripheral blood mononuclear cells, bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors. Incertain embodiments of the present invention, any number of T cell linesavailable and known to those skilled in the art, may be used. In anotherembodiment, said cell can be derived from a healthy donor, from apatient diagnosed with cancer or from a patient diagnosed with aninfection. In another embodiment, said cell is part of a mixedpopulation of cells which present different phenotypic characteristics.

In accordance with the present invention, the nucleic acid moleculesdetailed herein may be introduced in the T-cell by any suitable methodsknown in the art. Suitable, non-limiting methods for introducing anucleic acid molecule into a T-cell according include stabletransformation methods, wherein the nucleic acid molecule is integratedinto the genome of the cell, transient transformation methods whereinthe nucleic acid molecule is not integrated into the genome of the celland virus mediated methods. Said nucleic acid molecule may be introducedinto a cell by, for example, a recombinant viral vector (e.g.,retroviruses, adenoviruses), liposome and the like. Transienttransformation methods include, for example, microinjection,electroporation or particle bombardment. In certain embodiments, thenucleic acid molecule is a vector, such as a viral vector or plasmid.Suitably, said vector is an expression vector enabling the expression ofthe respective polypeptide(s) or protein(s) detailed herein by theT-cell.

A nucleic acid molecule introduced into the T-cell may be DNA or RNA. Incertain embodiments, a nucleic acid molecule introduced into the T-cellis DNA. In certain embodiments, a nucleic acid molecule introduced intothe T-cell is RNA, and in particular an mRNA encoding a polypeptide orprotein detailed herein, which mRNA is introduced directly into theT-cell, for example by electroporation. A suitable electroporationtechnique is described, for example, in International PublicationWO2013/176915 (in particular the section titled “Electroporation”bridging pages 29 to 30). A particular nucleic acid molecule which maybe an mRNA is the nucleic acid molecule comprising a nucleotide sequencecoding for a rare-cutting endonuclease able to selectively inactivate byDNA cleavage at least one gene coding for one component of the T-CellReceptor (TCR).

Peptide Inhibitor of FoxP3

The peptide inhibitor of FoxP3 in accordance with the present inventionmay be any peptide or polypeptide capable of inhibiting the activity ofthe forkhead/winged helix transcription factor 3 (FoxP3, preferablyhuman FoxP3), a transcription factor specific for regulatory T-cells andrequired for their development and function. With “inhibiting” is meantthat the activity of FoxP3 in regulatory T-cells is reduced by at least10%, such as at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80, at least 90%, at least 95%, atleast 99% or 100%. In this respect, “activity of FoxP3” meanstranscriptional activity.

Moreover, in addition to having a FoxP3 inhibitory activity, the peptideinhibitor of FoxP3 in accordance of the present invention, is capable ofpenetrating a cell membrane. This functionality may be inherent to thepeptide inhibitor of FoxP3 or may be the result of fusing a knowncell-penetrating peptide (CPP) and a peptide or polypeptide having FoxP3inhibitory activity. Said CPP sequence may be N-terminally orC-terminally linked to the amino acid sequence providing for the FoxP3inhibitory activity. Suitable examples for CPPs include, but are notlimited to: Tat, a nuclear transcriptional activator protein which is a101 amino acid protein required for viral replication by humanimmunodeficiency virus type 1 (HIV-1), penetratin, which corresponds tothe third helix of the homeoprotein Antennapedia in Drosophila, Kaposifibroblast growth factor (FGF) signal peptide sequence, integrin β3signal peptide sequence; Guanine rich-molecular transporters, MPG,pep-1, sweet arrow peptide, dermaseptins, transportan, pVEC, Humancalcitonin, mouse prion protein (mPrPr), polyarginine peptide Argssequence, VP22 protein from Herpes Simplex Virus, antimicrobial peptidesBuforin I and SynB (US2013/0065314).

A non-limiting example of peptide inhibitor of FoxP3 in accordance withthe present invention is the polypeptide P60 described by Casares et al.(2010). In addition to its FoxP3 inhibitory activity, it has been shownthat P60 is also able to penetrate a cell membrane. This polypeptide hasthe amino acid sequence: RDFQSFRKMWPFFAM [SEQ ID NO: 1]. An illustrativenucleotide sequence coding for this polypeptide is represented byCGCGACTTTCAAAGTTTCCGTAAGATGTGGCCGTTTTTTGCAATG [SEQ ID NO: 2]. However,it is understood that due to the degeneration of the genetic code anyother suitable nucleotide sequence coding for the amino acid sequenceset forth in SEQ ID NO: 1 is also encompassed by the present disclosure.

Accordingly, in certain embodiments of the invention, thecell-penetrating peptide inhibitor of FoxP3 is a polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 1 or a variant thereofcomprising an amino acid sequence that has at least 60%, such as atleast 80%, at least 85%, at least 90% or at least 95%, sequence identitywith the amino acid sequence set forth in SEQ ID NO: 1 over the entirelength of SEQ ID NO: 1. Hence, in accordance with these embodiments, anexogenous nucleic acid molecule comprising a nucleotide sequence codingfor a polypeptide comprising the amino acid sequence set forth in SEQ IDNO: 1 or a variant thereof comprising an amino acid sequence that has atleast 60%, such as at least 80%, at least 85%, at least 90% or at least95%, sequence identity with the amino acid sequence set forth in SEQ IDNO: 1 over the entire length of SEQ ID NO: 1 is introduced into theT-cell. The variant may comprise an amino acid sequence which has one ormore, such as two, three, four, five or six amino acid substitutionscompared to SEQ ID NO: 1. Preferably, such amino acid substitution is aconservative substitution which means that one amino acid is replaced byanother one that is similar in size and chemical properties. Suchconservative amino acid substitution may thus have minor effects on thepeptide structure and can thus be tolerated without compromisingfunction. Preferably, such variant is capable of inhibiting the activityof FoxP3 and is capable of penetrating a cell membrane.

In accordance with certain embodiments, the exogenous nucleic acidmolecule may thus comprise the nucleotide sequence set forth in SEQ IDNO: 2 or any other nucleotide sequence which due to the degeneration ofthe genetic code also codes for the amino acid sequence set forth in SEQID NO: 1.

As a result, an engineered T-cell may be obtained which expresses apolypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1or a variant thereof comprising an amino acid sequence that has at least60%, such as at least 80%, at least 85%, at least 90% or at least 95%,sequence identity with the amino acid sequence set forth in SEQ ID NO: 1over the entire length of SEQ ID NO: 1.

According to certain embodiments of the invention, the cell-penetratingpeptide inhibitor of FoxP3 is a polypeptide comprising the amino acidsequence MRDFQSFRKMWPFFAM [SEQ ID NO: 3] or a variant thereof comprisingan amino acid sequence that has at least 60%, such as at least 80%sequence identity with the amino acid sequence set forth in SEQ ID NO: 3over the entire length of SEQ ID NO: 3. Hence, in accordance with theseembodiments, an exogenous nucleic acid molecule comprising a nucleotidesequence coding for a polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 3 or a variant thereof comprising an amino acidsequence that has at least 60%, such as at least 62.5%, at least 75% orat least 87.5% sequence identity with the amino acid sequence set forthin SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 is introducedinto the T-cell. The polypeptide comprising an amino acid sequence thathas at least 60% sequence identify with the amino acid sequence setforth in SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 maycomprise an amino acid sequence which has one or more, such as two,three, four, five or six amino acid substitutions compared to SEQ ID NO:3. Preferably, such amino acid substitution is a conservativesubstitution which means that one amino acid is replaced by another onethat is similar in size and chemical properties.

Such conservative amino acid substitution may thus have minor effects onthe peptide structure and can thus be tolerated without compromisingfunction. Preferably, such variant is capable of inhibiting the activityof FoxP3 and is capable of penetrating a cell membrane.

In accordance with certain embodiments, the exogenous nucleic acidmolecule may thus comprise the nucleotide sequenceATGCGCGACTTTCAAAGTTTCCGTAAGATGTGG CCGTTTTTTGCAATG [SEQ ID NO: 4] or anyother nucleotide sequence which due to the degeneration of the geneticcode also codes for the amino acid sequence set forth in SEQ ID NO: 3.

As a result, an engineered T-cell may be obtained which expresses apolypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3or a variant thereof comprising an amino acid sequence that has at least60%, such as at least 62.5%, at least 75% or at least 87.5% sequenceidentity with the amino acid sequence set forth in SEQ ID NO: 3 over theentire length of SEQ ID NO: 3.

Chimeric Antigen Receptors (CARs)

Adoptive immunotherapy, which involves the transfer of autologousantigen-specific T-cells generated ex vivo, is a promising strategy totreat cancer or viral infections. The T-cells used for adoptiveimmunotherapy can be generated either by expansion of antigen-specific Tcells or redirection of T cells through genetic engineering (Park,Rosenberg et al. 2011). Transfer of viral antigen specific T-cells is awell-established procedure used for the treatment of transplantassociated viral infections and rare viral-related malignancies.Similarly, isolation and transfer of tumor specific T cells has beenshown to be successful in treating melanoma.

Novel specificities in T-cells have been successfully generated throughthe genetic transfer of transgenic T-cell receptors or chimeric antigenreceptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptorsconsisting of a targeting moiety that is associated with one or moresignaling domains in a single fusion molecule. In general, the bindingmoiety of a CAR consists of an antigen-binding domain of a single-chainantibody (scFv), comprising the light and variable fragments of amonoclonal antibody joined by a flexible linker. Binding moieties basedon receptor or ligand domains have also been used successfully. Thesignaling domains for first generation CARs are derived from thecytoplasmic region of the CD3zeta or the Fc receptor gamma chains. Firstgeneration CARs have been shown to successfully redirect T cellcytotoxicity, however, they failed to provide prolonged expansion andanti-tumor activity in vivo. Signaling domains from co-stimulatorymolecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have beenadded alone (second generation) or in combination (third generation) toenhance survival and increase proliferation of CAR modified T-cells.CARs have successfully allowed T-cells to be redirected against antigensexpressed at the surface of tumor cells from various malignanciesincluding lymphomas and solid tumors (Jena, Dotti et al. 2010).

CD19 is an attractive target for immunotherapy because the vast majorityof B-acute lymphoblastic leukemia (B-ALL) uniformly express CD19,whereas expression is absent on non hematopoietic cells, as well asmyeloid, erythroid, and T cells, and bone marrow stem cells. Clinicaltrials targeting CD19 on B-cell malignancies are underway withencouraging anti-tumor responses. Most infuse T cells geneticallymodified to express a chimeric antigen receptor (CAR) with specificityderived from the scFv region of a CD19-specific mouse monoclonalantibody FMC63 (WO2013/126712).

Therefore, in accordance with certain embodiments, the first ChimericAntigen Receptor is directed against the B-lymphocyte antigen CD19.

In accordance with certain embodiments, the first Chimeric AntigenReceptor is a single chain Chimeric Antigen Receptor. As an example ofsingle-chain Chimeric Antigen Receptor to be expressed in the engineeredT-cells according to the present invention is a single polypeptide thatcomprises at least one extracellular ligand binding domain, atransmembrane domain and at least one signal transducing domain, whereinsaid extracellular ligand binding domain comprises a scFV derived fromthe specific anti-CD19 monoclonal antibody 4G7. Once transduced into theT-cell, for instance by using retroviral or lentiviral transduction,this CAR contributes to the recognition of CD19 antigen present at thesurface of malignant B-cells involved in lymphoma or leukemia.

In accordance with particular embodiments, the first Chimeric AntigenReceptor is a polypeptide comprising the amino acid sequence forth inSEQ ID NO: 5 or a variant thereof comprising an amino acid sequence thathas at least 70%, such as at least 80%, at least 90%, at least 95%, orat least 99%, sequence identity with the amino acid sequence set forthin SEQ ID NO: 5 over the entire length of SEQ ID NO: 5. Preferably, thevariant is capable of binding CD19.

In accordance with other certain embodiments, the first Chimeric AntigenReceptor may be directed against another antigen expressed at thesurface of a malignant or infected cell, such as a cluster ofdifferentiation molecule, such as CD16, CD64, CD78, CD96, CLL1, CD116,CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surfaceantigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA),epithelial cell adhesion molecule (EpCAM), epidermal growth factorreceptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40,disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72,glycosphingolipids, glioma-associated antigen, β-human chorionicgonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF,prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53,prostein, PSMA, surviving and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22,insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, amajor histocompatibility complex (MHC) molecule presenting atumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromalantigens, the extra domain A (EDA) and extra domain B (EDB) offibronectin and the A1 domain of tenascin-C(TnC A1) and fibroblastassociated protein (fap); a lineage-specific or tissue specific antigensuch as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4,B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a majorhistocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17),multiple myeloma or lymphoblastic leukaemia antigen, such as oneselected from TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRCSD(UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROTP53708), and FCRL5 (UNIPROT Q68SN8). a virus-specific surface antigensuch as an HIV-specific antigen (such as HIV gp120); an EBV-specificantigen, a CMV-specific antigen, a HPV-specific antigen, a LasseVirus-specific antigen, an Influenza Virus-specific antigen as well asany derivate or variant of these surface antigens.

In other certain embodiments, the first Chimeric Antigen Receptor is amulti-chain Chimeric Antigen Receptor. Chimeric Antigen Receptors fromthe prior art introduced in T-cells have been formed of single chainpolypeptides that necessitate serial appending of signaling domains.However, by moving signaling domains from their natural juxtamembraneposition may interfere with their function. To overcome this drawback,the applicant recently designed a multi-chain CAR derived from FcεRI toallow normal juxtamembrane position of all relevant signaling domains.In this new architecture, the high affinity IgE binding domain of FcεRIalpha chain is replaced by an extracellular ligand-binding domain suchas scFv to redirect T-cell specificity against cell targets and the Nand/or C-termini tails of FcεRI beta chain are used to placecostimulatory signals in normal juxtamembrane positions as described inWO 2013/176916.

Accordingly, a CAR expressed by the genetically engineered T-cellaccording to the invention can be a multi-chain chimeric antigenreceptor particularly adapted to the production and expansion ofengineered T-cells of the present invention. Such multi-chain

CARs comprise at least two of the following components:

-   -   a) one polypeptide comprising the transmembrembrane domain of        FcεRI alpha chain and an extracellular ligand-binding domain,    -   b) one polypeptide comprising a part of N- and C-terminal        cytoplasmic tail and the transmembrane domain of FcεRI beta        chain and/or    -   c) at least two polypeptides comprising each a part of        intracytoplasmic tail and the transmembrane domain of FcεRI        gamma chain, whereby different polypeptides multimerize together        spontaneously to form dimeric, trimeric or tetrameric CAR.

According to such architectures, ligands binding domains and signalingdomains are born on separate polypeptides. The different polypeptidesare anchored into the membrane in a close proximity allowinginteractions with each other. In such architectures, the signaling andco-stimulatory domains can be in juxtamembrane positions (i.e. adjacentto the cell membrane on the internal side of it), which is deemed toallow improved function of co-stimulatory domains. The multi-subunitarchitecture also offers more flexibility and possibilities of designingCARs with more control on T-cell activation. For instance, it ispossible to include several extracellular antigen recognition domainshaving different specificity to obtain a multi-specific CARarchitecture. It is also possible to control the relative ratio betweenthe different subunits into the multi-chain CAR. This type ofarchitecture has been recently detailed by the applicant inPCT/US2013/058005.

The assembly of the different chains as part of a single multi-chain CARis made possible, for instance, by using the different alpha, beta andgamma chains of the high affinity receptor for IgE (FcεRI) (Metzger,Alcaraz et al. 1986) to which are fused the signaling and co-stimulatorydomains. The gamma chain comprises a transmembrane region andcytoplasmic tail containing one immunoreceptor tyrosine-based activationmotif (ITAM) (Cambier 1995).

The multi-chain CAR can comprise several extracellular ligand-bindingdomains, to simultaneously bind different elements in target therebyaugmenting immune cell activation and function. In one embodiment, theextracellular ligand-binding domains can be placed in tandem on the sametransmembrane polypeptide, and optionally can be separated by a linker.In another embodiment, said different extracellular ligand-bindingdomains can be placed on different transmembrane polypeptides composingthe multi-chain CAR.

The signal transducing domain or intracellular signaling domain of themulti-chain CAR(s) of the invention is responsible for intracellularsignaling following the binding of extracellular ligand binding domainto the target resulting in the activation of the immune cell and immuneresponse. In other words, the signal transducing domain is responsiblefor the activation of at least one of the normal effector functions ofthe immune cell in which the multi-chain CAR is expressed. For example,the effector function of a T cell can be a cytolytic activity or helperactivity including the secretion of cytokines.

In the present application, the term “signal transducing domain” refersto the portion of a protein which transduces the effector signalfunction signal and directs the cell to perform a specialized function.

Preferred examples of signal transducing domain for use in single ormulti-chain CAR can be the cytoplasmic sequences of the Fc receptor or Tcell receptor and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivate or variant of these sequences and any synthetic sequence thatas the same functional capability. Signal transduction domain comprisestwo distinct classes of cytoplasmic signaling sequence, those thatinitiate antigen-dependent primary activation, and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal. Primary cytoplasmic signaling sequence can comprise signalingmotifs which are known as immunoreceptor tyrosine-based activationmotifs of ITAMs. ITAMs are well defined signaling motifs found in theintracytoplasmic tail of a variety of receptors that serve as bindingsites for syk/zap70 class tyrosine kinases. Examples of ITAM used in theinvention can include as non-limiting examples those derived fromTCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon,CD5, CD22, CD79a, CD79b and CD66d. According to particular embodiments,the signaling transducing domain of the multi-chain CAR can comprise theCD3zeta signaling domain, or the intracytoplasmic domain of the FcεRIbeta or gamma chains.

According to particular embodiments, the signal transduction domain ofmulti-chain CARs of the present invention comprises a co-stimulatorysignal molecule. A co-stimulatory molecule is a cell surface moleculeother than an antigen receptor or their ligands that is required for anefficient immune response.

Ligand binding-domains can be any antigen receptor previously used, andreferred to, with respect to single-chain CAR referred to in theliterature, in particular scFv from monoclonal antibodies.

In accordance with particular embodiments, the first Chimeric AntigenReceptor is a polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 6 (encoded by, e.g., SEQ ID NO: 7) or a variant thereofcomprising an amino acid sequence that has at least 70%, such as atleast 80%, at least 90%, at least 95%, or at least 99%, sequenceidentity with the amino acid sequence set forth in SEQ ID NO: 6 over theentire length of SEQ ID NO: 6. Preferably, the variant is capable ofbinding CD19.

A particularly preferred first Chimeric Antigen Receptor is apolypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8or a variant thereof comprising an amino acid sequence that has at least80%, such as at least 90%, at least 95%, or at least 99%, sequenceidentity with the amino acid sequence set forth in SEQ ID NO: 8 over theentire length of SEQ ID NO: 8. Preferably, said variant is capable ofbinding CD19.

Also encompassed by the present invention are bispecific ormulti-specific CARs as described, for instance, in InternationalPublication WO 2014/4011988. Such bi-specific or multi-specific CARs areparticularly contemplated with respect to the second CAR which isdirected against at least one antigen expressed at the surface of aregulatory T-cell.

A suitable target antigen for the second CAR is the surface antigenCD25, which is known to be expressed on the surface of regulatoryT-cells. This will allow the binding of a regulatory T-cell by theengineered T-cell of the invention. Other exemplary surface antigens maybe CD4, CD152, IL3R, CCR4, CCR6, CD161 and CXR3.

According to certain embodiments, the second Chimeric Antigen Receptoris mono-specific and, preferably, is directed against surface antigenCD25.

According to other certain embodiments, the second Chimeric AntigenReceptor is bi-specific. According to particular embodiments, suchbi-specific second Chimeric Antigen Receptor is directed against surfaceantigen CD25 and one other surface antigen selected from the groupconsisting of CD4, CD152, IL3R, CCR4, CCR6, CD161 and CXR3.

As with the first Chimeric Antigen Receptor, the second Chimeric AntigenReceptor may be a single chain Chimeric Antigen Receptor or amulti-chain CAR. The details provided above with respect to the singlechain and multi-chain CARs apply mutatis mutandis.

Activation and Expansion of T-Cells

Whether prior to or after genetic modification of the T-cell(s), theT-cell(s) can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005. The T-cell(s) may be expanded in vitro or in vivo.

Generally, the T-cell(s) of the invention is expanded by contact with asurface having attached thereto an agent that stimulates a CD3 TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T-cellpopulations may be stimulated in vitro such as by contact with ananti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2antibody immobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of theT-cells, a ligand that binds the accessory molecule is used. Forexample, a T-cell or population of T-cells can be contacted with ananti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T-cells.

In further embodiments of the present invention, the T-cells, arecombined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. Cell surface proteinsmay be ligated by allowing paramagnetic beads to which anti-CD3 andanti-CD28 are attached (3×28 beads) to contact the T cells. According toone embodiment, the cells (for example, 4 to 10 T-cells) and beads (forexample, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of1:1) are combined in a buffer, preferably PBS (without divalent cationssuch as, calcium and magnesium). Again, those of ordinary skill in theart can readily appreciate any cell concentration may be used. Themixture may be cultured for several hours (about 3 hours) to about 14days or any hourly integer value in between. In another embodiment, themixture may be cultured for 21 days. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, 1L-4,1L-7, GM-CSF, -10, -2, 1L-15, TGFp, and TNF- or any other additives forthe growth of cells known to the skilled artisan. Other additives forthe growth of cells include, but are not limited to, surfactant,plasmanate, and reducing agents such as N-acetyl-cysteine and2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM,F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodiumpyruvate, and vitamins, either serum-free or supplemented with anappropriate amount of serum (or plasma) or a defined set of hormones,and/or an amount of cytokine(s) sufficient for the growth and expansionof T cells. Antibiotics, e.g., penicillin and streptomycin, are includedonly in experimental cultures, not in cultures of cells that are to beinfused into a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% C02). T cells that havebeen exposed to varied stimulation times may exhibit differentcharacteristics.

In another particular embodiment, the T-cells may be expanded byco-culturing with tissue or cells. Said T-cells may also be expanded invivo, for example in the subject's blood after administrating said cellinto the subject.

Engineered T-Cells

As a result of the present invention, engineered T-cells can be obtainedhaving improved characteristics. In particular, the present inventionprovides an engineered, preferably isolated, T-cell which ischaracterized by the expression of both a Chimeric Antigen Receptor(CAR) directed against at least one antigen expressed at the surface ofa malignant or infected cell, herein denoted first CAR, and an inhibitorof regulatory T-cell activity, preferably a cell-penetrating peptideinhibitor of FoxP3.

More particularly, the present invention provides an engineered,preferably isolated, T-cell which comprises:

a) an exogenous nucleic acid molecule comprising a nucleotide sequencecoding for a first Chimeric Antigen Receptor (CAR) directed against atleast one antigen expressed at the surface of a malignant or infectedcell; and

b) an exogenous nucleic acid molecule comprising a nucleotide sequencecoding for an inhibitor of regulatory T-cell activity, preferably acell-penetrating peptide inhibitor of FoxP3.

According to certain embodiments, the engineered T-cell furthercomprises c) an exogenous nucleic acid molecule comprising a nucleotidesequence coding for a second Chimeric Antigen Receptor directed againstat least one antigen expressed at the surface of a regulatory T-cell(Treg). According to particular embodiments, said second ChimericAntigen Receptor is expressed by said T-cell.

According to certain embodiments, the engineered T-cell furthercomprises d) an exogenous nucleic acid molecule comprising a nucleotidesequence coding for a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage at least one gene coding for one component ofthe T-Cell receptor (TCR). According to particular embodiments, saidrare-cutting endonuclease able to selectively inactivate by DNA cleavageat least one gene coding for one component of the T-Cell receptor (TCR)is expressed by said T-cell.

According to certain embodiments, the engineered T-cell furthercomprises e) an exogenous nucleic acid molecule comprising a nucleotidesequence coding for a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage the gene coding for the surface antigen CD25.According to particular embodiments, said rare-cutting endonuclease ableto selectively inactivate by DNA cleavage the gene coding for thesurface antigen CD25 is expressed by said T-cell.

It is understood that the details given herein in particularly withrespect to the first Chimeric Antigen Receptor, the inhibitor ofregulatory T-cell activity, especially the cell-penetrating peptideinhibitor of FoxP3, the second Chimeric Antigen Receptor, therare-cutting endonuclease able to selectively inactivate by DNA cleavageat least one gene coding for one component of the T-Cell receptor (TCR)and the rare-cutting endonuclease able to selectively inactivate by DNAcleavage the gene coding for the surface antigen CD25 also apply to thisaspect of the invention.

Further, in the scope of the present invention is also encompassed acell line obtained from a genetically engineered T-cell according to theinvention.

Nucleic Acids, Compositions and Kits

In further aspects, the present invention provides nucleic acidmolecules suitable for expressing the various CARs, the inhibitor ofregulatory T-cell activity, especially the peptide inhibitors FoxP3, andendonucleases in a T-cell as well as compositions and kits comprisingsuch nucleic acid molecules.

Accordingly, the present invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence coding for a Chimeric AntigenReceptor (CAR) directed against at least one antigen expressed at thesurface of a malignant or infected cell; and a nucleotide sequencecoding for an inhibitor of regulatory T-cell activity, preferably acell-penetrating peptide inhibitor of FoxP3.

The nucleic acid molecule may be DNA or RNA. In certain embodiments, thenucleic acid molecule is DNA. In certain other embodiments, the nucleicacid molecule is RNA molecule, and in particular an mRNA encoding saidChimeric Antigen Receptor and said cell-penetrating peptide inhibitor ofFoxP3. In accordance with particular embodiments, the nucleotidesequence coding for said Chimeric Antigen Receptor (CAR) and thenucleotide sequence coding for an inhibitor of regulatory T-cellactivity, preferably a cell-penetrating peptide inhibitor of FoxP3, areoperatively linked to each other by a nucleotide sequence coding forribosomal skip sequence, such as a nucleotide sequence coding for a 2Apeptide. Such ribosomal skip mechanisms are well known in the art andare known to be used by several vectors for the expression of severalproteins encoded by a single mRNA.

In accordance with certain embodiments, the nucleic acid is a vector,such as a viral vector or plasmid. In order to allow expression by theT-cell the nucleotide sequence coding for said Chimeric Antigen Receptor(CAR) and the nucleotide sequence coding for an inhibitor of regulatoryT-cells activity, preferably a cell-penetrating peptide inhibitor ofFoxP3, are operatively linked to one or more promoters suitable forexpression in a T-cell. In some cases it may be desirable to have theinhibitor of regulatory T-cells activity, such as the cell-penetratingpeptide inhibitor of FoxP3, only expressed if the Chimeric AntigenReceptor recognizes and binds the antigen to which it is specific. Insuch cases, the expression of the cell-penetrating peptide inhibitor ofFoxP3 is preferably under the control of an inducible promoter, such asa NFAT minimal promoter.

Also encompassed within the scope of the invention are compositionswhich comprise one or more of the nucleic acid molecules detailedherein. Particularly, the present invention provides compositionscomprising one or more nucleic acid molecules comprising a nucleotidesequence coding for a first Chimeric Antigen Receptor (CAR) directedagainst at least one antigen expressed at the surface of a malignant orinfected cell; and a nucleotide sequence coding for a cell-penetratingpeptide inhibitor of FoxP3. In accordance with certain embodiments, acomposition is provided which comprises a nucleic acid moleculecomprising a nucleotide sequence coding for said first Chimeric AntigenReceptor (CAR); and a nucleotide sequence coding for said an inhibitorof regulatory T-cell activity, preferably said cell-penetrating peptideinhibitor of FoxP3. In accordance with other certain embodiments, acomposition is provided which comprises a first nucleic acid moleculecomprising a nucleotide sequence coding for said first Chimeric AntigenReceptor (CAR) directed against at least one antigen expressed at thesurface of a malignant or infected cell; and a second nucleic acidmolecule comprising a nucleotide sequence coding for an inhibitor ofregulatory T-cell activity, preferably said cell-penetrating peptideinhibitor of FoxP3. In accordance with particular embodiments, thecomposition may comprise a further nucleic acid molecule comprising anucleotide sequence coding for a second Chimeric Antigen Receptordirected against at least one antigen expressed at the surface of aregulatory T-cell (Treg). In accordance with other particularembodiments, composition may comprise a further nucleic acid moleculecomprising a nucleotide sequence coding for a rare-cutting endonucleaseable to selectively inactivate by DNA cleavage at least one gene codingfor one component of the T-Cell receptor (TCR) and/or a nucleic acidmolecule comprising a nucleotide sequence coding for a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage the genecoding for the surface antigen CD25.

The nucleic acid molecule(s) comprised by the compositions may be DNA orRNA. In certain embodiments, a nucleic acid molecule is DNA. In certainother embodiments, a nucleic acid molecule is RNA molecule. Inaccordance certain embodiments, the nucleic acid molecule or nucleicacid molecules are vectors, such as viral vectors or plasmids. In orderto allow expression by the T-cell the nucleotide sequence coding forsaid Chimeric Antigen Receptor (CAR) and/or the nucleotide sequencecoding for an inhibitor of regulatory T-cell activity, preferably acell-penetrating peptide inhibitor of FoxP3, are operatively linked toone or more promoters suitable for expression in a T-cell.

Also encompassed within the scope of the invention are kits comprisingone or more of the nucleic acid molecules or one or more compositionsdetailed herein.

It is understood that the details given herein in particularly withrespect to the first Chimeric Antigen Receptor, the inhibitor ofregulatory T-cell activity, especially the cell-penetrating peptideinhibitor of FoxP3, the second Chimeric Antigen Receptor, therare-cutting endonuclease able to selectively inactivate by DNA cleavageat least one gene coding for one component of the T-Cell receptor (TCR)and the rare-cutting endonuclease able to selectively inactivate by DNAcleavage the gene coding for the surface antigen CD25 also apply tothese aspects of the invention.

Therapeutic Applications

The T-cells obtainable in accordance with the present invention areintended to be used as a medicament, and in particular for treating,among others, cancer, infections (such viral infections) or immunediseases in a patient in need thereof. Accordingly, the presentinvention provides engineered T-cells for use as a medicament.Particularly, the present invention provides engineered T-cells for usein the treatment of a cancer, such as lymphoma, or viral infection. Alsoprovided are compositions, particularly pharmaceutical compositions,which comprise at least one genetically engineered T-cell of the presentinvention. In certain embodiments, a composition may comprise apopulation of engineered T-cell of the present invention.

The treatment can be ameliorating, curative or prophylactic. It may beeither part of an autologous immunotherapy or part of an allogenicimmunotherapy treatment. By autologous, it is meant that cells, cellline or population of cells used for treating patients are originatingfrom said patient or from a Human Leucocyte Antigen (HLA) compatibledonor. By allogeneic is meant that the cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

The invention is particularly suited for allogenic immunotherapy,insofar as it enables the transformation of T-cells, typically obtainedfrom donors, into non-alloreactive cells. This may be done understandard protocols and reproduced as many times as needed. The resultedmodified T-cells may be pooled and administrated to one or severalpatients, being made available as an “off the shelf” therapeuticproduct.

The treatments are primarily to treat patients diagnosed with cancer.Cancers are preferably leukemias and lymphomas, which have liquidtumors, but may also concern solid tumors. Types of cancers to betreated with the genetically engineered T-cells of the inventioninclude, but are not limited to, carcinoma, blastoma, and sarcoma, andcertain leukemia or lymphoid malignancies, benign and malignant tumors,and malignancies e.g., sarcomas, carcinomas, and melanomas. Adulttumors/cancers and pediatric tumors/cancers are also included.

The treatment can take place in combination with one or more therapiesselected from the group of antibodies therapy, chemotherapy, cytokinestherapy, dendritic cell therapy, gene therapy, hormone therapy, laserlight therapy and radiation therapy.

According to certain embodiments, T-cells of the invention can undergorobust in vivo T-cell expansion upon administration to a patient, andcan persist in the body fluids for an extended amount of time,preferably for a week, more preferably for 2 weeks, even more preferablyfor at least one month. Although the T-cells according to the inventionare expected to persist during these periods, their life span into thepatient's body are intended not to exceed a year, preferably 6 months,more preferably 2 months, and even more preferably one month.

The administration of the cells or population of cells according to thepresent invention may be carried out in any convenient manner, includingby aerosol inhalation, injection, ingestion, transfusion, implantationor transplantation. The compositions described herein may beadministered to a patient subcutaneously, intradermaliy, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous orintralymphatic injection, or intraperitoneally. In one embodiment, thecell compositions of the present invention are preferably administeredby intravenous injection.

The administration of the cells or population of cells can consist ofthe administration of 104-109 cells per kg body weight, preferably 105to 106 cells/kg body weight including all integer values of cell numberswithin those ranges. The cells or population of cells can beadministrated in one or more doses. In another embodiment, saideffective amount of cells are administrated as a single dose. In anotherembodiment, said effective amount of cells are administrated as morethan one dose over a period time. Timing of administration is within thejudgment of managing physician and depends on the clinical condition ofthe patient. The cells or population of cells may be obtained from anysource, such as a blood bank or a donor. While individual needs vary,determination of optimal ranges of effective amounts of a given celltype for a particular disease or conditions within the skill of the art.An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administrated will be dependent uponthe age, health and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment and the nature of the effectdesired.

In other embodiments, said effective amount of cells or compositioncomprising those cells are administrated parenterally. Saidadministration can be an intravenous administration. Said administrationcan be directly done by injection within a tumor.

In certain embodiments, cells are administered to a patient inconjunction with (e.g., before, simultaneously or following) any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as antiviral therapy, cidofovir and interleukin-2,Cytarabine (also known as ARA-C) or nataliziimab treatment for MSpatients or efaliztimab treatment for psoriasis patients or othertreatments for PML patients. In further embodiments, the T cells of theinvention may be used in combination with chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin,mycoplienolic acid, steroids, FR901228, cytokines, and irradiation.These drugs inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin) (Liu et al., Cell66:807-815, 1 1; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Citrr. Opin. mm n. 5:763-773, 93). In a further embodiment, thecell compositions of the present invention are administered to a patientin conjunction with (e.g., before, simultaneously or following) bonemarrow transplantation, T cell ablative therapy using eitherchemotherapy agents such as, fludarabine, external-beam radiationtherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH,In another embodiment, the cell compositions of the present inventionare administered following B-cell ablative therapy such as agents thatreact with CD20, e.g., Rituxan. For example, in one embodiment, subjectsmay undergo standard treatment with high dose chemotherapy followed byperipheral blood stem cell transplantation. In certain embodiments,following the transplant, subjects receive an infusion of the expandedgenetically engineered T-cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

Also encompassed within this aspect of the invention are methods fortreating a patient in need thereof, comprising a) providing at least oneengineered T-cell of the present invention, preferably a population ofsaid T-cell; and b) administering said T-cell or population to saidpatient.

Also encompassed within this aspect of the invention are methods forpreparing a medicament using at least one engineered T-cell of thepresent invention, and preferably a population of said T-cell.Accordingly, the present invention provides the use of at least oneengineered T-cell of the present invention, and preferably a populationof said T-cell, in the manufacture of a medicament. Preferably, suchmedicament is for use in the treatment of a cancer, such as lymphoma, orviral infection.

Other Definitions

-   -   Amino acid residues in a polypeptide sequence are designated        herein according to the one-letter code, in which, for example,        Q means Gln or Glutamine residue, R means Arg or Arginine        residue and D means Asp or Aspartic acid residue.    -   Amino acid substitution means the replacement of one amino acid        residue with another, for instance the replacement of an        Arginine residue with a Glutamine residue in a peptide sequence        is an amino acid substitution.    -   Nucleotides are designated as follows: one-letter code is used        for designating the base of a nucleoside: a is adenine, t is        thymine, c is cytosine, and g is guanine. For the degenerated        nucleotides, r represents g or a (purine nucleotides), k        represents g or t, s represents g or c, w represents a or t, m        represents a or c, y represents t or c (pyrimidine nucleotides),        d represents g, a or t, v represents g, a or c, b represents g,        t or c, h represents a, t or c, and n represents g, a, t or c.    -   “As used herein, “nucleic acid” or “polynucleotides” refers to        nucleotides and/or polynucleotides, such as deoxyribonucleic        acid (DNA) or ribonucleic acid (RNA), oligonucleotides,        fragments generated by the polymerase chain reaction (PCR), and        fragments generated by any of ligation, scission, endonuclease        action, and exonuclease action. Nucleic acid molecules can be        composed of monomers that are naturally-occurring nucleotides        (such as DNA and RNA), or analogs of naturally-occurring        nucleotides (e.g., enantiomeric forms of naturally-occurring        nucleotides), or a combination of both. Modified nucleotides can        have alterations in sugar moieties and/or in pyrimidine or        purine base moieties. Sugar modifications include, for example,        replacement of one or more hydroxyl groups with halogens, alkyl        groups, amines, and azido groups, or sugars can be        functionalized as ethers or esters. Moreover, the entire sugar        moiety can be replaced with sterically and electronically        similar structures, such as aza-sugars and carbocyclic sugar        analogs. Examples of modifications in a base moiety include        alkylated purines and pyrimidines, acylated purines or        pyrimidines, or other well-known heterocyclic substitutes.        Nucleic acid monomers can be linked by phosphodiester bonds or        analogs of such linkages. Nucleic acids can be either single        stranded or double stranded.    -   By “delivery vector” or “delivery vectors” is intended any        delivery vector which can be used in the present invention to        put into cell contact (i.e “contacting”) or deliver inside cells        or subcellular compartments (i.e “introducing”) agents/chemicals        and molecules (proteins or nucleic acids) needed in the present        invention. It includes, but is not limited to liposomal delivery        vectors, viral delivery vectors, drug delivery vectors, chemical        carriers, polymeric carriers, lipoplexes, polyplexes,        dendrimers, microbubbles (ultrasound contrast agents),        nanoparticles, emulsions or other appropriate transfer vectors.        These delivery vectors allow delivery of molecules, chemicals,        macromolecules (genes, proteins), or other vectors such as        plasmids, or penetrating peptides. In these later cases,        delivery vectors are molecule carriers.    -   The terms “vector” or “vectors” refer to a nucleic acid molecule        capable of transporting another nucleic acid to which it has        been linked. A “vector” in the present invention includes, but        is not limited to, a viral vector, a plasmid, a RNA vector or a        linear or circular DNA or RNA molecule which may consists of a        chromosomal, non-chromosomal, semi-synthetic or synthetic        nucleic acids. Preferred vectors are those capable of autonomous        replication (episomal vector) and/or expression of nucleic acids        to which they are linked (expression vectors). Large numbers of        suitable vectors are known to those of skill in the art and        commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e.g.adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies andvesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai),positive strand RNA viruses such as picornavirus and alphavirus, anddouble-stranded DNA viruses including adenovirus, herpesvirus (e.g.,Herpes Simplex virus types 1 and 2, Epstein-Barr virus,cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses,papovavirus, hepadnavirus, and hepatitis virus, for example. Examples ofretroviruses include: avian leukosis-sarcoma, mammalian C-type, B-typeviruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin,J. M., Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields, et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996).

-   -   By “lentiviral vector” is meant HIV-Based lentiviral vectors        that are very promising for gene delivery because of their        relatively large packaging capacity, reduced immunogenicity and        their ability to stably transduce with high efficiency a large        range of different cell types. Lentiviral vectors are usually        generated following transient transfection of three (packaging,        envelope and transfer) or more plasmids into producer cells.        Like HIV, lentiviral vectors enter the target cell through the        interaction of viral surface glycoproteins with receptors on the        cell surface. On entry, the viral RNA undergoes reverse        transcription, which is mediated by the viral reverse        transcriptase complex. The product of reverse transcription is a        double-stranded linear viral DNA, which is the substrate for        viral integration in the DNA of infected cells. By “integrative        lentiviral vectors (or LV)”, is meant such vectors as non        limiting example, that are able to integrate the genome of a        target cell. At the opposite by “non integrative lentiviral        vectors (or NILV)” is meant efficient gene delivery vectors that        do not integrate the genome of a target cell through the action        of the virus integrase.    -   Delivery vectors and vectors can be associated or combined with        any cellular permeabilization techniques such as sonoporation or        electroporation or derivatives of these techniques.    -   By cell or cells is intended any eukaryotic living cells,        primary cells and cell lines derived from these organisms for in        vitro cultures. Preferably, the cell or cells are human cells.    -   By “primary cell” or “primary cells” are intended cells taken        directly from living tissue (i.e. biopsy material) and        established for growth in vitro, that have undergone very few        population doublings and are therefore more representative of        the main functional components and characteristics of tissues        from which they are derived from, in comparison to continuous        tumorigenic or artificially immortalized cell lines.    -   by “mutation” is intended the substitution, deletion, insertion        of up to one, two, three, four, five, six, seven, eight, nine,        ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty        five, thirty, fourty, fifty, or more nucleotides/amino acids in        a polynucleotide (cDNA, gene) or a polypeptide sequence. The        mutation can affect the coding sequence of a gene or its        regulatory sequence. It may also affect the structure of the        genomic sequence or the structure/stability of the encoded mRNA.    -   by “variant(s)”, it is intended a repeat variant, a variant, a        DNA binding variant, a TALE-nuclease variant, a polypeptide        variant obtained by mutation or replacement of at least one        residue in the amino acid sequence of the parent molecule.    -   by “functional variant” is intended an active mutant of a        protein, polypeptide or a protein domain; such mutant may have        the same activity compared to its parent protein, polypeptide or        protein domain or additional properties, or higher or lower        activity.    -   By “gene” is meant the basic unit of heredity, consisting of a        segment of DNA arranged in a linear manner along a chromosome,        which codes for a specific protein or segment of protein. A gene        typically includes a promoter, a 5′ untranslated region, one or        more coding sequences (exons), optionally introns, a 3′        untranslated region. The gene may further comprise a terminator,        enhancers and/or silencers.    -   The term “cleavage” refers to the breakage of the covalent        backbone of a polynucleotide. Cleavage can be initiated by a        variety of methods including, but not limited to, enzymatic or        chemical hydrolysis of a phosphodiester bond. Both        single-stranded cleavage and double-stranded cleavage are        possible, and double-stranded cleavage can occur as a result of        two distinct single-stranded cleavage events. Double stranded        DNA, RNA, or DNA/RNA hybrid cleavage can result in the        production of either blunt ends or staggered ends.    -   By “fusion protein” is intended the result of a well-known        process in the art consisting in the joining of two or more        genes which originally encode for separate proteins or part of        them, the translation of said “fusion gene” resulting in a        single polypeptide with functional properties derived from each        of the original proteins.    -   “identity” refers to sequence identity between two nucleic acid        molecules or polypeptides. Identity can be determined by        comparing a position in each sequence which may be aligned for        purposes of comparison. When a position in the compared sequence        is occupied by the same base or amino acid, then the molecules        are identical at that position. A degree of similarity or        identity between nucleic acid or amino acid sequences is a        function of the number of identical or matching nucleotides or        amino acids at positions shared by the nucleic acid or amino        acid sequences, respectively. Various alignment algorithms        and/or programs may be used to calculate the identity between        two sequences, including FASTA, or BLAST which are available as        a part of the GCG sequence analysis package (University of        Wisconsin, Madison, Wis.), and can be used with, e.g., default        setting. For example, polypeptides having at least 70%, 85%,        90%, 95%, 98% or 99% identity to specific polypeptides described        herein and preferably exhibiting substantially the same        functions, as well as polynucleotide encoding such polypeptides,        are contemplated.    -   “signal-transducing domain” or “co-stimulatory ligand” refers to        a molecule on an antigen presenting cell that specifically binds        a cognate co-stimulatory molecule on a T-cell, thereby providing        a signal which, in addition to the primary signal provided by,        for instance, binding of a TCR/CD3 complex with an MHC molecule        loaded with peptide, mediates a T cell response, including, but        not limited to, proliferation activation, differentiation and        the like. A co-stimulatory ligand can include but is not limited        to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L,        inducible costimulatory igand (ICOS-L), intercellular adhesion        molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB,        HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist        or antibody that binds Toll ligand receptor and a ligand that        specifically binds with B7-H3. A co-stimulatory ligand also        encompasses, inter alia, an antibody that specifically binds        with a co-stimulatory molecule present on a T cell, such as but        not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS,        lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,        LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.    -   A “co-stimulatory molecule” refers to the cognate binding        partner on a Tcell that specifically binds with a co-stimulatory        ligand, thereby mediating a co-stimulatory response by the cell,        such as, but not limited to proliferation. Co-stimulatory        molecules include, but are not limited to an MHC class I        molecule, BTLA and Toll ligand receptor.    -   A “co-stimulatory signal” as used herein refers to a signal,        which in combination with primary signal, such as TCR/CD3        ligation, leads to T cell proliferation and/or upregulation or        downregulation of key molecules.    -   “bispecific antibody” refers to an antibody that has binding        sites for two different antigens within a single antibody        molecule. It will be appreciated by those skilled in the art        that other molecules in addition to the canonical antibody        structure may be constructed with two binding specificities. It        will further be appreciated that antigen binding by bispecific        antibodies may be simultaneous or sequential. Bispecific        antibodies can be produced by chemical techniques (see e.g.,        Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78, 5807), by        “polydoma” techniques (See U.S. Pat. No. 4,474,893) or by        recombinant DNA techniques, which all are known per se. As a        non-limiting example, each binding domain comprises at least one        variable region from an antibody heavy chain (“VH or H region”),        wherein the VH region of the first binding domain specifically        binds to the lymphocyte marker such as CD3, and the VH region of        the second binding domain specifically binds to tumor antigen.    -   The term “extracellular ligand-binding domain” as used herein is        defined as an oligo- or polypeptide that is capable of binding a        ligand. Preferably, the domain will be capable of interacting        with a cell surface molecule. For example, the extracellular        ligand-binding domain may be chosen to recognize a ligand that        acts as a cell surface marker on target cells associated with a        particular disease state. Thus examples of cell surface markers        that may act as ligands include those associated with viral,        bacterial and parasitic infections, autoimmune disease and        cancer cells.    -   The term “subject” or “patient” as used herein includes all        members of the animal kingdom including non-human primates and        humans.

The above written description of the invention provides a manner andprocess of making and using it such that any person skilled in this artis enabled to make and use the same, this enablement being provided inparticular for the subject matter of the appended claims, which make upa part of the original description.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

EXAMPLES Example 1: Treg Inhibition Test

Human primary T cells were activated with anti-CD3/CD28 beads. At day 3,they were transfected with a messenger RNA coding for foxp3 inhibitorypeptide p60 (SEQ ID NO: 1) fused to a mutated Chicken lysozyme signalpeptide (3R CLSP—SEQ ID NO: 12) (alternative SEQ ID NO. 13 to 17displayed in Table 1 may also be used). At day four, the transfected Tcells were mixed with human regulatory T cells (Treg) and theirproliferation was followed according to the assay described by Collison,L. W et al. (In vitro Treg suppression assays, Methods Mol. Biol., 2011,707:21-37). This assay shows that the T-cells transfected with the p60messenger RNAs, proliferate faster than those transfected with the mockRNA (scrambled p60—SEQ ID NO: 9), upon contact with the regulatory Tcells. It resulted that p60 peptide expression allowed T cells to resistTreg inhibition.

Example 2: Cytotoxic Activity Test

Human primary T cells is activated with anti-CD3/CD28 beads. At day 3,the activated T cells are transduced with a lentiviral vector encodingthe chimeric antigen receptor anti-CD19 set forth as SEQ ID NO: 5 alongwith the Foxp3 inhibitory peptide p60 (SEQ ID NO: 1) fused to a mutatedChicken lysozyme signal peptide (3R CLSP—SEQ ID NO: 12) (alternative SEQID NO. 13 to 17 displayed in Table 1 may also be used). At day 5, thecytotoxic activity of the transduced T cells are assayed according tothe method described by Yang, Z. Z. et al. (Attenuation of CD8(+) T cellfunction by CD4(+)CD25(+)regulatory T cells in B-cell non-Hodgkin'slymphoma, 2006, Cancer Res.) against a relevant target cell line in thepresence or absence of Tregs.

The assay shows that regulatory T cells usually have an inhibitoryeffect on the cytotoxic capacity of CAR+ T cells, whereas p60 peptideexpression by the T cell restores cytotoxic activity by lifting thisinhibition.

TABLE 1 Sequences used in the examples Polypeptide Amino acid sequenceSEQ ID NO # p60 RDFQSFRKMWPFFAM SEQ ID NO: 1 control (scrambled p60)MKMFFDAFPQRRSWF SEQ ID NO: 9 linker GSSSS SEQ ID NO: 10Chicken lysozyme signal peptide (CLSP) MRSLLILVLCFLPLAALG SEQ ID NO: 113R CLSP MRRRSLLILVLCFLPLAALG SEQ ID NO: 12 4R CLSP MRRRRSLLILVLCFLPLAALGSEQ ID NO: 13 Human lysozyme signal peptide (HSLP) MKALIVLGLVLLSVTVQGSEQ ID NO: 14 Human interleukin 2 signal peptide (IL2SP)MYRMQLLSCIALSLALVTNS SEQ ID NO: 15 3K HLSP MKKKALIVLGLVLLSVTVQGSEQ ID NO: 16 3R IL2SP MYRRRMQLLSCIALSLALVTNS SEQ ID NO: 17

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1. A method for preparing an engineered T-cell comprising the steps of: a) providing a T-cell; b) introducing into said T-cell an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a first Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell; and c) introducing into said T-cell an exogenous nucleic acid molecule comprising a nucleotide sequence coding for an inhibitor of regulatory T-cell activity.
 2. The method according to claim 1, wherein the inhibitor of regulatory T-cell activity is an inhibitor of FoxP3.
 3. The method according to claim 2, wherein the inhibitor of FoxP3 is a cell penetrating peptide.
 4. The method according to any one of claims 1 to 3, further comprising the step of: d) introducing into said T-cell an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a second Chimeric Antigen Receptor directed against at least one antigen expressed at the surface of a regulatory T-cell (Treg).
 5. The method according to any one of claims 1 to 4, further comprising the step of: e) introducing into said T-cell an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a rare-cutting endonuclease able to selectively inactivate by DNA cleavage at least one gene coding for one component of the T-Cell receptor (TCR).
 6. The method according to any one of claims 1 to 5, further comprising the step of: f) introducing into said T-cell an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a rare-cutting endonuclease able to selectively inactivate by DNA cleavage the gene coding for the surface antigen CD25.
 7. The method according to any one of claims 1 to 6, further comprising the step of: g) expanding the resulting engineered T-cell.
 8. The method according to any one of claims 3 to 7, wherein the cell-penetrating peptide inhibitor of FoxP3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof comprising an amino acid sequence that has at least 60%, such as at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 over the entire length of SEQ ID NO:
 1. 9. The method according to any one of claims 1 to 8, wherein the first Chimeric Antigen Receptor is directed against the B-lymphocyte antigen CD19.
 10. The method according to any one of claims 1 to 8, wherein the first Chimeric Antigen Receptor is directed against an antigen selected from a cluster of differentiation molecule, such as CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C (TnC A1) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), multiple myeloma or lymphoblastic leukaemia antigen, such as one selected from TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRCSD (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), and FCRL5 (UNIPROT Q68SN8), a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen as well as any derivate or variant of these surface antigens.
 11. The method according to any one of claims 1 to 10, wherein the first or second Chimeric Antigen Receptor is a single chain Chimeric Antigen Receptor.
 12. The method according to any one of claims 1 to 10, wherein the first or second Chimeric Antigen Receptor is a multi-chain Chimeric Antigen Receptor.
 13. The method according to any one of claims 4 to 13, wherein the second Chimeric Antigen Receptor is directed against surface antigen CD25.
 14. The method according to any one of claims 4 to 13, wherein the second Chimeric Antigen Receptor is directed against a first surface antigen which is CD25 and a second surface antigen which is selected from the group consisting of CD4, CD152, IL3R, CCR4, CCR6, CD161 and CXR3.
 15. The method according to any one of claims 5 to 14, wherein the rare-cutting endonuclease according to e) selectively inactivates the gene coding for TCR alpha or TCR beta.
 16. The method according to any one of claims 1 to 15, wherein said T-cell is derived from a cytotoxic T-lymphocyte.
 17. An engineered, preferably isolated, T-cell comprising: a) an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a first Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell; and b) an exogenous nucleic acid molecule comprising a nucleotide sequence coding for an inhibitor of regulatory T-cell activity.
 18. The engineered T-cell according to claim 17, wherein the inhibitor of regulatory T-cell activity is an inhibitor of FoxP3.
 19. The engineered T-cell according to claim 18, wherein the inhibitor of FoxP3 is a cell penetrating peptide.
 20. The engineered T-cell according to any one of claims 17 to 19, wherein said first Chimeric Antigen Receptor and said inhibitor of regulatory T-cell activity are expressed by said T-cell.
 21. The engineered T-cell according to any one of claims 17 to 20, further comprising: c) an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a second Chimeric Antigen Receptor directed against at least one antigen expressed at the surface of a regulatory T-cell (Treg).
 22. The engineered T-cell according to claim 21, wherein said second Chimeric Antigen Receptor is expressed by said T-cell.
 23. The engineered T-cell according to any one of claims 17 to 22, further comprising: d) an exogenous nucleic acid molecule comprising a nucleotide sequence coding for a rare-cutting endonuclease able to selectively inactivate by DNA cleavage at least one gene coding for one component of the T-Cell receptor (TCR).
 24. The engineered T-cell according to any one of claims 17 to 23, wherein said cell further comprises a deletion or a mutation into at least one gene coding for one component of the T-Cell receptor (TCR).
 25. The engineered T-cell according to any one of claims 19 to 24, wherein the cell-penetrating peptide inhibitor of FoxP3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a variant thereof comprising an amino acid sequence that has at least 60%, such as at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 over the entire length of SEQ ID NO:
 1. 26. The engineered T-cell according to any one of claims 17 to 25, wherein the first Chimeric Antigen Receptor is directed against the B-lymphocyte antigen CD19.
 27. The engineered T-cell according to any one of claims 17 to 25, wherein the first Chimeric Antigen Receptor is directed against an antigen selected from a cluster of differentiation molecule, such as CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the A1 domain of tenascin-C(TnC A1) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), multiple myeloma or lymphoblastic leukaemia antigen, such as one selected from TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), and FCRL5 (UNIPROT Q68SN8), a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen as well as any derivate or variant of these surface antigens.
 28. The engineered T-cell according to any one of claims 17 to 27, wherein the first or second Chimeric Antigen Receptor is a single chain Chimeric Antigen Receptor.
 29. The engineered T-cell according to any one of claims 17 to 27, wherein the first or second Chimeric Antigen Receptor is a multi-chain Chimeric Antigen Receptor.
 30. The engineered T-cell according to any one of claims 17 to 29, wherein the second Chimeric Antigen Receptor is mono-specific.
 31. The engineered T-cell according to any one of claims 17 to 29, wherein the second Chimeric Antigen Receptor is multi-specific.
 32. The engineered T-cell according to any one of claims 17 to 31, wherein the second Chimeric Antigen Receptor is directed against surface antigen CD25.
 33. The engineered T-cell according to claim 31, wherein the second Chimeric Antigen Receptor is directed against a first surface antigen which is CD25 and a second surface antigen which is selected from the group consisting of CD4, CD152, IL3R, CCR4, CCR6, CD161 and CXR3.
 34. The engineered T-cell according to any one of claims 17 to 33, wherein said T-cell expresses a rare-cutting endonuclease that selectively inactivates the gene coding for TCR alpha or TCR beta.
 35. The engineered T-cell according to any one of claims 17 to 34, wherein said T-cell is derived from a cytotoxic T-lymphocyte.
 36. The engineered T-cell according to any one of claims 17 to 35 for use as a medicament.
 37. The engineered T-cell according to any one of claims 17 to 35 for use in the treatment of a cancer or viral infection.
 38. The engineered T-cell according to any one of claims 17 to 35 for use in the treatment of lymphoma.
 39. A composition comprising at least one genetically engineered T-cell according to any one of claims 17 to
 35. 40. An isolated nucleic acid molecule comprising a nucleotide sequence coding for a Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell; and a nucleotide sequence coding for an inhibitor of regulatory T-cell activity, such as a cell-penetrating peptide inhibitor of FoxP3.
 41. The isolated nucleic acid according to claim 40, wherein said nucleotide sequence coding for said Chimeric Antigen Receptor (CAR) and said nucleotide sequence coding for an inhibitor of regulatory T-cell activity are operatively linked to each other by a nucleotide sequence coding for ribosomal skip sequence, such as a nucleotide sequence coding for a 2A peptide.
 42. The isolated nucleic acid according to claim 40 or 41, which is a vector, such as a viral vector or plasmid, and said nucleotide sequences being operatively linked to one or more promoters suitable for expression in a T-cell.
 43. A composition comprising one or more nucleic acid molecules comprising a nucleotide sequence coding for a first Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell; and a nucleotide sequence coding for an inhibitor of regulatory T-cell activity, such as a cell-penetrating peptide inhibitor of FoxP3.
 44. The composition according to claim 43, comprising a nucleic acid molecule comprising a nucleotide sequence coding for said first Chimeric Antigen Receptor (CAR); and a nucleotide sequence coding for said inhibitor of regulatory T-cell activity.
 45. The composition according to claim 43, comprising a first nucleic acid molecule comprising a nucleotide sequence coding for said first Chimeric Antigen Receptor (CAR) directed against at least one antigen expressed at the surface of a malignant or infected cell; and a second nucleic acid molecule comprising a nucleotide sequence coding for said inhibitor of regulatory T-cell activity.
 46. The composition according to any one of claims 43 to 45, comprising a further nucleic acid molecule comprising a nucleotide sequence coding for a second Chimeric Antigen Receptor directed against at least one antigen expressed at the surface of a regulatory T-cell (Treg).
 47. The composition according to any one of claims 43 to 46, wherein the nucleic acid molecules are vectors, such as viral vectors or plasmids, and said nucleotide sequences being operatively linked to one or more promoters suitable for expression in a T-cell.
 48. A kit comprising the isolated nucleic acid according to any one of claims 40 to 42 or a composition according to any one of claims 43 to
 47. 